Three new species of Pseudorhabdosynochus (Monogenea, Diplectanidae) from several species of Cephalopholis and Epinephelus (Perciformes, Serranidae) from Thailand

Pseudorhabdosynochus suratthaniensis n. sp. is described from the gills of Cephalopholis argus; P. cephalopholi n. sp., from the gills of C. sonnerati; and P. samaesarnensis n. sp., from the gills of Epinephelus lanceolatus. These fish were all caught in the Gulf of Thailand. Pseudorhabdosynochus suratthaniensis n. sp. is distinguished from congeneric species by the structure of its sclerotized vagina, which has a wide sclerotized trumpet and a single large primary chamber, and by the number of rows of rodlets in each of its squamodiscs. Pseudorhabdosynochus cephalopholi n. sp. is also distinguished by the structure of its sclerotized vagina that, like the P. suratthaniensis n. sp., has a sclerotized trumpet, but it also has a long coiled or curved primary canal near its midlength, and a distal part with a primary chamber and a secondary chamber communicating with the primary chamber through a short secondary canal. In addition, P. cephalopholi n. sp. is distinguished by some sclerotized organs (ventral and dorsal hamuli, ventral bar, and quadriloculate organ) with different lengths, and by the number of rows of rodlets in each of its squamodiscs. Pseudorhabdosynochus samaesarnensis n. sp. is distinguished by its sclerotized vagina that has an anterior cup-shaped trumpet and a short straight or curved primary canal. For Thailand, these are the first species of Pseudorhabdosynochus described from species of Cephalopholis and the second species of Pseudorhabdosynochus described from Epinephelus.


Introduction
Groupers (Serranidae, Epinephelinae) are bottom-associated fishes found in tropical and subtropical seas. Most species occur in coral reefs, but some live in estuaries or on rocky reefs [11]. The Epinephelinae are divided into five tribes that comprise about 234 species of marine fishes in 32 genera [28]. The hybrid grouper, a cross of the female tiger grouper E. fuscoguttatus and the male giant grouper E. lanceolatus (tiger grouper Â giant grouper or TGGG), has taken the Asian aquaculture industry by storm since 2006. TGGG is widely cited as the most successful hybrid combination, as it is able to grow quickly [2,6]. In Thailand, the peacock hind Cephalopholis argus (Bloch & Schneider), the tomato hind C. sonnerati (Valenciennes), the coral hind C. miniata (Forsskål), the giant grouper Epinephelus lanceolatus (Bloch), and the whitespotted grouper E. coeruleopunctatus (Bloch) occur in coral reefs and other coastal and offshore areas; the malabar grouper E. malabaricus (Bloch & Schneider) occurs in mangroves; the cloudy grouper E. erythrurus (Valenciennes) occurs in seagrass beds; and the orange-spotted grouper E. coioides (Hamilton) occurs in both mangroves and seagrass beds [29]. Species of Pseudorhabdosynochus Yamaguti, 1958 are mostly found on the gills of groupers mainly of the genus Epinephelus and appear to be specific to their host [18,22]. Pseudorhabdosynochus currently has 96 valid species [10]. Four of those species -P. argus Justine, 2007, P. minutus Justine, 2007, P. urceolus Mendoza-Franco, Violante-González & Herrera, 2011, and P. meganmarieae Kritsky, Bakenhaster & Adams, 2015have been described from four species of Cephalopholis -C. argus, C. sonnerati, C. panamensis (Steindachner), and C. cruentata (Lacepède) [15,24,26], respectively. In this paper, we describe three new species -P. suratthaniensis n. sp. found on the gills of C. argus, P. cephalopholi n. sp. found on the gills of C. sonnerati, and P. samaesarnensis n. sp. found on the gills of E. lanceolatusin the Gulf of Thailand. Pseudorhabdosynochus argus and P. minutus had earlier been described from C. argus and C. sonnerati off Nouméa, New Caledonia [15], respectively; in this paper, we describe a second species of Pseudorhabdosynochus from these fish. In addition, we describe the first species of Pseudorhabdosynochus from E. lanceolatus: Pseudorhabdosynochus samaesarnensis n. sp.

Materials and methods
One specimen of C. argus (total length, 303 mm and weight, 443 g) was obtained from a jetty in Surat Thani province, Southern Thailand in June 2019. Four specimens of C. sonnerati (total length, 230-247 mm and weight, 234-287 g), and nine specimens of C. miniata (total length, 210-340 mm and weight, 169-637 g) were obtained from a jetty in Surat Thani province, Southern Thailand in May 2019. One specimen of E. lanceolatus (total length, 520 mm and weight, 5000 g) was obtained from a local fisherman on Samaesarn Island in the Gulf of Thailand, Chonburi province, Eastern Thailand in September 2020. Four specimens of E. coeruleopunctatus (total length, 290-330 mm and weight, 378-557 g), four specimens of E. coioides (total length, 350-470 mm and weight, 617-1500 g), five specimens of E. erythrurus (total length, 245-275 mm and weight, 260-338 g), four specimens of E. malabaricus (total length, 360-370 mm and weight, 759-844 g), and eleven specimens of E. fuscoguttatus (total length, 250-350 mm and weight, 270-914 g) were obtained from a local fisherman on Libong Island in the Andaman Sea, Trang province, Southern Thailand in April 2020. Three sea cage-cultured hybrid groupers (TGGG) (E. fuscoguttatus $ Â E. lanceolatus #) (total length, 380-480 mm and weight, 1100-2600 g) were obtained from a local farmer in Ban Laem Hin, Phang-Nga province in the Andaman Sea, Southern Thailand in May 2020. All the fish were dead and were immediately transported in a cool box to the laboratory. Their gills were removed and placed in Petri dishes that contained seawater. Monogeneans were individually picked off the gills with a fine needle with the aid of a stereomicroscope and put on slides. They were prepared with ammonium picrate-glycerin, referred to as "Picrate" (see [13]) according to [25]. Then, their soft internal organs and haptoral hard parts were immediately examined. The slides were later sealed with Canada balsam [13]. Specimens were photographed using an Olympus DP 70 microscope (Olympus Corporation, Japan) and a Zeiss Axiocam 506 color microscope (Carl Zeiss AG, Germany) for drawing. Various sclerotized organs were measured based on a previous study (see Fig. 1 in [13,14]; their nomenclature follows [14]). Measurements are in lm and given as mean followed by (minimum-maximum, n=) between parentheses. Site in host: Gills. Infection indices: Prevalence 100% (one specimen examined and infected); 36 helminth specimens on the single grouper examined.
Etymology: The species name "suratthaniensis" is treated as an adjective and was derived from the name of the province "Surat Thani", where the host fish Cephalopholis argus was collected.

Differential diagnosis
Pseudorhabdosynochus suratthaniensis n. sp. is easily distinguished from other species of Pseudorhabdosynochus by the structure of its sclerotized vagina and the number of rows of rodlets in each of its squamodiscs. Another Pseudorhabdosynochus species that has a vaginal structure similar to that of P. suratthaniensis n. sp. is P. urceolus from C. panamensis from Taboga Island in Panama. The general structure of the sclerotized vagina appeared to be similar to that of the P. suratthaniensis n. sp. Pseudorhabdosynochus urceolus can be distinguished by the following characteristics: the size of its sclerotized vagina (29 versus 40 lm in P. suratthaniensis n. sp.); the morphology of its sclerotized vagina, with a bell-shaped opening that is not in P. suratthaniensis n. sp; and its chamber structure, with a small hollow structure on its margin that is not in P. suratthaniensis n. sp. In addition, the squamodiscs of P. urceolus have numerous rows of rodlets, that is, 14-15 rows of rodlets and a 0-1 innermost row that form complete concentric rings; but the squamodiscs of P. suratthaniensis n. sp. have 8-11 rows of rodlets and 1-2 innermost rows that form closed ovals [26]. Pseudorhabdosynochus bouaini Neifar & Euzet, 2007 from E. costae (Steindachner) out of Sfax, Tunisia has a sclerotized vagina similar to that of P. suratthaniensis n. sp. It is characterized by an anterior trumpet, followed by a short and heavily sclerotized primary canal progressively in the heavy primary chamber. However, the distal part of the primary chamber has two small sclerotized protuberances in P. bouaini (which are not in P. suratthaniensis n. sp) [27].
Other host: C. miniata (Forsskål) (Perciformes, Serranidae) Type locality: Surat Thani Province, the lower Gulf of Thailand, Southern Thailand (9°48 Etymology: Species name derived from Cephalopholis, which is the name of the host genus (both type-host and other host). This monogenean species seems to be specific to this host genus. Sclerotized vagina with a complex sclerotized structure, aspect changes according to specimen and orientation (Figs. 4A-4I). Sclerotized vagina comprises anterior trumpet, followed by primary canal, primary chamber, secondary canal, secondary chamber, and accessory structure; trumpet in continuity with primary canal tube, long, coiled (Fig. 4F), or curved (Figs. 4A-E and 4G-4I) at around midlength, heavily sclerotized, progressively into primary chamber or form a bend in posterior region and progressively into primary chamber (Fig. 4H); secondary chamber communicates with primary chamber through a short secondary canal, and accessory structure connected to secondary chamber. Total length of sclerotized vagina (measured from distal extremity of trumpet to base of vagina, not considering curved length along coil or curve of primary canal) 25 (19-30, n = 25), length variable because of variation in coil or curvature of primary canal.

Differential diagnosis
Pseudorhabdosynochus cephalopholi n. sp. is distinguished from other Pseudorhabdosynochus species by the structure of its sclerotized vagina, in addition to the differing lengths of some sclerotized organs and the number of rows of rodlets in each squamodisc. Pseudorhabdosynochus minutus from C. sonnerati out of the Barrier reef off Nouméa, New Caledonia has the following characteristics. The general morphology of its sclerotized vagina and its size (25 lm), body size (270-550 lm versus 260-645 lm in P. cephalopholi n. sp.), number of rows of rodlets in each squamodisc (10-11 rows of rodlets and a 0-1 closed oval), and host species (C. sonnerati) are similar to those of P. cephalopholi n. sp. Pseudorhabdosynochus minutus can be distinguished, however, by the chamber structure of its sclerotized vagina (P. minutus has spherical chambers, unlike the chambers in P. cephalopholi n. sp.). Pseudorhabdosynochus argus from C. argus out of the Barrier reef off Nouméa, New Caledonia has a sclerotized vagina similar to that of P. cephalopholi n. sp., characterized by an anterior trumpet followed by a long primary canal. Pseudorhabdosynochus argus can be distinguished, however, by the size of its sclerotized vagina (38 lm vs. 25 lm in P. cephalopholi n. sp.); the morphology of its sclerotized vagina, with the primary canal coiled in its anterior part, just after the trumpet (see [15]), unlike in P. cephalopholi n. sp.; its body size (888 lm versus C. Saengpheng and W. Purivirojkul: Parasite 2022, 29, 48   (Table 1). Pseudorhabdosynochus sp. Justine, 2007 from C. boenak (Bloch) out of Heron Island, Queensland, Australia has a sclerotized vagina similar to that of P. cephalopholi n. sp., characterized by an anterior trumpet followed by a long primary canal. Pseudorhabdosynochus sp. can be distinguished by the following: the morphology of its primary canal, the shapes of its chambers, and the number of rows of rodlets in each of its squamodiscs (7 rows of rodlets, including 0-2 closed rows versus 8-10 rows of rodlets and an innermost row closed oval in P. cephalopholi n. sp.). Pseudorhabdosynochus sp. generally resembles a dwarf P. argus [15]. Pseudorhabdosynochus euitoe Justine, 2007 from E. maculatus (Bloch) out of the Barrier reef off Nouméa, New Caledonia has a sclerotized vagina similar to that of P. cephalopholi n. sp., characterized by an anterior trumpet followed by a long, coiled primary canal. However, the trumpet is characterized by a heavily sclerotized ring at the trumpetcanal limit in the P. euitoe (which is absent in P. cephalopholi n. sp.). Pseudorhabdosynochus fuitoe Justine, 2007 from E. maculatus out of the Barrier reef off Nouméa, New Caledonia has a sclerotized vagina similar to that of P. cephalopholi n. sp., characterized by an anterior trumpet followed by a heavy, long, coiled or curved primary canal, but the shapes of their chambers differ [14]. Pseudorhabdosynochus manifestus Justine & Sigura, 2007 from E. malabaricus (Bloch & Schneider) out of the Lagoon off Nouméa, New Caledonia is differentiated by its sclerotized vagina and its cone-shaped primary canal that is coiled in its anterior part and straight in its posterior part (which is absent in P. cephalopholi n. sp.) [20].  Site in host: Gills. Infection indices: Prevalence 100% (one specimen examined and infected); 12 helminth specimens on the single grouper examined.
Etymology: The species name "samaesarnensis" was derived from "Samaesarn Island", where the host fish Epinephelus lanceolatus was collected.

Description (Figs. 5 and 6)
[Based on 6 specimens]. Body (including haptor) 745 long (679-821, n = 6); maximum width 198 (160-234, n = 6). Tegument scaly (observed in some specimens). Anterior region with Table 1. Measurements of P. cephalopholi n. sp. from the type host, C. sonnerati, C. miniata, and two previously described Pseudorhabdosynochus species from Cephalopholis spp. The measurements of P. cephalopholi n. sp. were taken from flattened specimens in ammonium picrate-glycerin. Sclerotized vagina with a complex sclerotized structure, aspect changes according to specimen and orientation (Figs. 6A-6F). Sclerotized vagina comprises anterior cupshaped trumpet, followed by primary canal, primary chamber and accessory structure; thick-walled anterior cup-shaped trumpet in continuity with short straight or curved primary canal (Fig. 6A), thick-walled sclerotization and widens into a thickwalled primary chamber; accessory structure connects to the primary chamber. Total length of sclerotized vagina (measured from distal extremity of trumpet to base of vagina, not considering curve along primary canal) 27 (25-29, n = 6), length variable because of variation in curvature of primary canal.

Differential diagnosis
Pseudorhabdosynochus samaesarnensis n. sp. is distinguished from other Pseudorhabdosynochus species by the structure of its sclerotized vagina. Pseudorhabdosynochus samaesarnensis n. sp. has a vaginal structure similar to that of P. nhatrangensis from E. coioides (Hamilton) and E. bleekeri (Vaillant) off Vietnam. The structure of their sclerotized vagina was found to be very similar, but the number of rows of rodlets found in P. nhatrangensis was always 10 [7], while in P. samaesarnensis n. sp., the rows were found to vary in number from 9 to 13. In addition, the specimens of P. samaesarnensis n. sp. from E. lanceolatus were larger than those of P. nhatrangensis from E. coioides and E. bleekeri (Table 2). However, P. nhatrangensis Dang, Bristow, Schander & Berland, 2013 is not a valid species because the article was not compliant with the new Article 8.5.3 of the International Code of Zoological Nomenclature (ICZN) [3].
Three new species of Pseudorhabdosynochus are described. Pseudorhabdosynochus suratthaniensis n. sp. was found on a single host species, C. argus, while P. cephalopholi n. sp. and P. samaesarnensis n. sp. were found on two host species and seven host species, respectively. Pseudorhabdosynochus cephalopholi n. sp. was found on C. sonnerati and C. miniata, and its morphometric data from both C. sonnerati and C. miniata are similar in size (Table 1). In addition, the structures of the sclerotized vaginae are no different (P. cephalopholi n. sp., which is rare in C. miniata). Pseudorhabdosynochus samaesarnensis n. sp. was found on seven host species: E. lanceolatus, E. coioides, E. erythrurus, E. coeruleopunctatus, E. malabaricus, sea cage-cultured hybrid grouper (TGGG) (E. fuscoguttatus $ Â E. lanceolatus #), and E. fuscoguttatus, and the morphometric data of P. samaesarnensis n. sp. from the six hosts are similar in size (Table 3). In addition, the structures of the sclerotized vaginae are no different. This table does not include the details of P. samaesarnensis n. sp. from E. fuscoguttatus because its specimens are incomplete and are found in low numbers (prevalence: about 9.1%). Pseudorhabdosynochus samaesarnensis n. sp. has a structure of the sclerotized vagina different from other species previously found in host fish -E. coioides [1,9,[37][38][39], E. erythrurus [30], E. coeruleopunctatus [35], E. malabaricus [20], and sea cage-cultured hybrid    [8]. We hypothesized that P. cephalopholi n. sp. and P. samaesarnensis n. sp. have low specificity to their hosts and that their infestations of more than one congeneric host species help to perpetuate this parasitic species [36].